The catalytic mechanism and unique low pH optimum ofCaldicellulosiruptor besciifamily 3 pectate lyase

Abstract

The unique active site of theCaldicellulosiruptor besciifamily 3 pectate lyase (PL3) enzyme has been thoroughly characterized using a series of point mutations, X-ray crystallography, pKacalculations and biochemical assays. The X-ray structures of seven PL3 active-site mutants, five of them in complex with intact trigalacturonic acid, were solved and characterized structurally, biochemically and computationally. The results confirmed that Lys108 is the catalytic base, but there is no clear candidate for the catalytic acid. However, the reaction mechanism can also be explained by an antiperiplanartrans-elimination reaction, in which Lys108 abstracts a proton from the C5 atom without the help of simultaneous proton donation by an acidic residue. An acidified water molecule completes theantiβ-elimination reaction by protonating the O4 atom of the substrate. Both the C5 hydrogen and C4 hydroxyl groups of the substrate must be orientated in axial configurations, as for galacturonic acid, for this to be possible. The wild-typeC. besciiPL3 displays a pH optimum that is lower than that ofBacillus subtilisPL1 according to activity measurements, indicating thatC. besciiPL3 has acquired a lower pH optimum by utilizing lysine instead of arginine as the catalytic base, as well as by lowering the pKaof the catalytic base in a unique active-site environment.